Arg-Gly-Asp (RGD) ligand clustering has been implicated in the modulation of cell adhesion, cell migration, cell spreading, and non-viral gene transfer [1-5]. For example, the maximum distance RGD ligands can be spaced for cell attachment and migration to occur on a flat surface is 58 nm [6] and it has been shown that integrin occupancy, conformation, and aggregation regulate integrin-signal transduction [7,8]. Interestingly, four different adenovirus sterotypes [9], have evolved to take advantage of clustered RGD ligands to gain access into cells through binding multiple integrin receptors simultaneously [10]. The clustered RGD ligands are displayed on five penton base proteins located at each of the twelve vertices of the virus surface. The penton base protein protrudes five RGD peptide sequences located 5.7 nm apart [9, 11], which have been found to be critical for viral cell entry [12]. Inhibition of the penton base protein using antibodies, RGD peptides, or by mutation results in decreased adenoviral internalization and overall transduction efficiency [11, 13-15].
The introduction of multivalent ligand binding to drug delivery carriers has been investigated as a method to enhance delivery of small molecular drugs [16] or tumor labeling agents [17] and has been demonstrated to have increased effects over monovalent binding [18-21]. However, the introduction of clustered ligand binding to non-viral gene delivery vectors has not been investigated. Nevertheless, ligands have been introduced to the surface of non-viral vectors to enhance targetability and overall gene transfer efficiency. These ligands include small molecules (e.g. folate and galactose), proteins (e.g transferrin and antibodies) [22] as well as RGD peptides [23, 24]. Although the direct RGD conjugation to DNA/poly(ethylene imine) (PEI) polyplexes increases the transfection efficiency in vitro and in vivo [23-25], the effect of RGD ligand clustering or the clustering of any other ligand on the efficiency and targetability of DNA/PEI polyplexes has not been investigated to date.
The cationic polymer PEI is one of the most widely used non-viral gene delivery vehicles for DNA, being used successfully both in vitro and in vivo [26]. It condenses with DNA through its positively charged amines, protecting DNA from degradation and forming particles (polyplexes) that can enter the cell and result in transgene expression [26]. The amines in PEI also serve as functional groups for chemical modification to introduce domains such as poly(ethylene glycol) to increase biocompatibility [27, 28], and ligands and peptides that enhance targeting, internalization and trafficking [29].